DESCRIPTION (from applicant's abstract): Opioid peptides such as enkephalins and endorphins are formed through the action of processing enzymes known as prohormone convertase. Studies funded by this proposal are directed toward a basic understanding of the cell biology and biochemistry of the interaction of these convertases with their neuropeptide precursor substrates in neuroendocrine cells. Our past work has addressed the production and characterization of recombinant sources of enzymes and substrates, and has established cell culture models in which the action of each convertase- as well as convertase inhibitors-can be separately studied. Using proenkephalin as a model substrate, we have demonstrated that PC1 and PC2 exhibit distinct specificity preferences, with PC2 the actual enzyme responsible for the production of bioactive opioids. We now propose to complete these studies by performing mass spectroscopy, radioimmunoassay, and western blotting to identify all major cleavage sites of proenkephalin by each enzyme; further, we will extend our specificity studies to the new opioid precursor, pronociceptin. We will detail the molecular basis for specificity differences between PC1 and PC2 by performing site-directing mutagenesis of PC2 to systematically investigate the individual pocket residues potentially conferring specificity. Additionally, in collaboration with two skilled peptide chemists, Drs. Richard Houghten and Dr. Luiz Juliano, we propose to develop synthetic inhibitors of these enzymes, using combinatorial chemistry on the one hand, and structure-function analysis of the only known endogenous inhibitor, the 7B2CT peptide, as another approach. Lastly, for in vivo work, we will design variious inhibitor-containing expressing vector constructs to target secretory vesicles, the major site of production of PC2 derived opioids. These constructs will be based upon two distinct strategies, alternatively using the CT peptide or the PC propeptide as potential inhibitory agents. Taken together, our proposed experiments to control the molecular interactions of processing enzymes with their opioid precursor substrates both in vitro as well as in vivo is expected to yield interesting new results generally applicable to the study of neuropeptide and peptide hormone biosynthesis.